1. Field of the Invention
The present invention relates to improved methods of delivering radioisotopes to tumor cells for effecting targeted radioimmunotherapy (RAIT). More specifically, the present invention relates to a radioimmunoconjugate wherein an alpha- or beta-emitting radioisotope is complexed to a binding agent attached to a fragment of an immunoglobulin such as Fabxe2x80x2. A clearing agent such as D-lysine may be administered to the patient along with the radioimmunoconjugate so that targeted tumor or cancer cells are destroyed, but damage to organs and tissues is minimized. After administering a radioimmunoconjugate to a patient, bone marrow or autologous stem-cells may be grafted to the patient.
2. Related Art
One therapeutic method used in cancer treatment involves directing antibodies carrying a therapeutic agent or cytotoxic compound to the diseased tissues. When localized at the disease site, the antibody delivers the therapeutic agent or cytotoxic compound to the cancerous cells. One approach to this methodology involves delivering radioisotopes to the diseased cells. This approach has proven useful in diagnosis where a radioisotope with particular imaging properties is delivered to the targeted diseased tissue.
Several methods have been used in radioimmunotherapy (RAIT). In one method, a radioisotope with desirable properties is carried by an antibody to a diseased tissue with a corresponding antigen. Various immunoglobulins such as IgG and IgM have been used to carry radioisotopes to an antigen located on a targeted disease tissue.
Various radioisotopes have been used in RAIT. 212Bi and its parent 212Pb have been successfully chelated to antibodies and other proteins via DTPA (diethylenetriamine pentaacetic acid) and DOTA (tetraazacyclododecane-N,Nxe2x80x2,Nxe2x80x3, Nxe2x80x2xe2x80x3-tetraacetic acid) derivatives and used as alpha emitting radioisotopes in RAIT. For example, Macklis et al. disclose a radioimmunoconjugate directed against a murine antigen present on the surface of malignant T-cells. Science, 240, 1024, 1988. The radioimmunoconjugate disclosed by Macklis et al. includes a 212Bi complexed to a cyclic anhydride of DTPA attached to a monoclonal antibody. Science, 240, 1024, 1988. One drawback to the use of 212Bi and its parent 212Pb in RAIT is that the decay product of 212Bi is 209Tl which is a high-energy xcex2xe2x88x92 and xcex3-emitter. This characteristic raises radiation protection problems.
Another alpha-emitting radioisotope that has been investigated in immunotherapy is 212At. Bloomer et al. Science, 212, 340, 1988. The half-life of 211At of 7.2 hours is significantly longer than that of 212Bi which has a half-life of only 60.55 minutes. It is known that 212At undergoes 42% alpha decay and 58% electron capture. The alpha particle emitted upon decay has an energy of 5.94 MeV whereas electron capture produces 80 keV of xcex3 radiation. The 211Po daughter arising from electron capture has a half-life of 0.5 seconds and decays by emitting an alpha particle with an energy of 7.43 MeV. Thus, by either rotute one obtains one high energy alpha particle per 211At decay. Isotopically pure 211At is accessible by electron capture of 211Rn which can be isolated in radiochemically pure form from irradiated thorium by gas chromatography. Chalkin et al. Chemiker-Ztg., 101, 470, 1977.
Astatine is a halogen atom and thus behaves similarly to iodine. It has long been known that blood clearance of 212At is very rapid because the astatine is quickly accumulated in the thyroid gland. Hamilton et al. Proc. Nat""l. Acad. Sci., 26, 483, 1940. In fact, studies performed on rats and monkeys in the 1950""s confirmed that the concentration of astatine in the thyroid is at least two orders of magnitude higher than in other organs, except for the stomach. Hamilton et al. Univ. Calif. Publ. Pharmacol., 2, 283, 1954.
Another radioisotope that has been investigated in conjunction with RAIT is 213Bi. This radioisotope is known to decay mainly (98%) by xcex2xe2x88x92 and 440 keV xcex3 emission with a half-life of 45.6 minutes to the ultra-short lived high-energy (8.375 MeV) alpha-emitter 213Po (txc2xd of 4 xcexcs), whereas a direct alpha-decay pathway to 209Tl plays only a negligible role (2% of all 213Bi decays).
U.S. Pat. No. 5,641,471 issued to Geerlings discloses a method for preparing 213Bi for therapeutic use. In the disclosed method a monoclonal antibody is used as a targeting moiety. A chelator such as CHX-DTPA (cyclohexyldiethylenetriamine pentaacetic acid) is attached to the antibody and functions to chelate the radioisotope. In this manner, the radioisotope is delivered to the target cell where it can function in a therapeutic manner to destroy the diseased tissue.
U.S. Pat. No. 5,246,691 issued to Geerlings et al. discloses radioimmunotherapy using alpha particle emissions. Specifically disclosed, is the use of 225Ac and its daughters as part of an immnunoconjugate also comprising an antibody such as human monoclonal antibody and humanized antibodies. The cited patent discloses that chelating agents are used to bind actinium and bismuth radioisotopes in the radioimmunoconjugate comprising a radionuclide that emits alpha particles, a chelating agent, and a slowly localizing antibody such as a human IgM antibody. Also disclosed is the use of scavenging agents such as DTPA, EDTA, PLED, and crown ethers for use in binding wandering isotopes and thus prohibiting the isotopes from invading non-targeted organs and tissues.
U.S. Pat. No. 5,428,154 issued to Gansow et al. discloses a chelate comprising a DOTA derivative and a metal including Pb, Bi, Y, and the lanthanides. Also disclosed is the linking of the metal chelate to a biomolecule to form a delivery system for the chelated metal. Gansow et al. discloses that both alpha and beta-emitters (212Pb, 212Bi, and 90Y) can be chelated to a DOTA derivative attached to a monoclonal antibody directed against an epitope on tumor cells.
A number of references have described radioimmunoconjugates comprising a radioisotope, and a chelator attached to a monoclonal antibody. However, a need remains for a radioimmunoconjugate that utilizes a Fabxe2x80x2 fragment of an immunoglobulin to target diseased tissue or cancer cells. A need also remains for a method of treating a patient that minimizes the amount of damage done by unbound radioisotopes.
It is an object of the present invention to provide a radioimmunoconjugate including a cytotoxic radioisotope bound to a binding site linked to or on an antigen-binding fragment of an antibody which specifically binds to a tumor-associated antigen.
Another object of the invention is to provide a method of treating a patient comprising administering to a patient a radioimmunoconjugate capable of targeting a diseased cell or tissue and delivering a radioisotope to the targeted cell or tissue.
Still another object of the invention is to provide a method of treating a patient that minimizes damage to non-targeted organs and tissues.
It is still another object of the invention to provide a method of treating a patient comprising administering a clearing agent to the patient in conjunction with a radioimmunoconjugate.
It is another object of the invention to provide a kit for use in radioimmunotherapy that includes a molecule with a radioisotope binding site linked to or on an antigen-binding fragment of an antibody which specifically binds to a tumor-associated antigen, and at least one clearing agent.
The present invention relates to radioimmunoconjugates including a cytotoxic radioisotope bound to a binding site linked to or on an antigen-binding fragment of an antibody which specifically binds to a tumor-associated antigen.
In preferred embodiments of the invention, the antibody is an IgG. In still other preferred embodiments of the invention, the fragment is a Fabxe2x80x2 fragment. In especially preferred embodiments, the Fabxe2x80x2 fragment is human, humanized or chimeric.
In preferred embodiments of the invention, the radioisotope of the radioimmunoconjugate is an alpha or beta emitter. More preferred embodiments include an alpha emitting radioisotope, and in the other preferred embodiments tile radioisotope of the radioimmunoconjugate is 213Bi, 90Y, or 211At.
In other preferred embodiments of the invention, the binding site of the radioimmnunoconjugate is selected from the group consisting of DTPA, DOTA, EDTA, PLED, a crown ether, an aromatic compound, and the sulflydryl group of a cysteine residue on a Fabxe2x80x2 fragment.
The present invention also relates to methods for treating a patient using the radioimmunoconjugate of the present invention.
In one embodiment, a method for treating a patient comprises administering any of the radioimmunoconjugates of the present invention to a patient. Other preferred embodiments comprise administering one or more clearing agents to a patient in conjunction with the administration of a radioimmunoconjugate. In more preferred embodiments, the clearing agent or agents are selected from the group consisting of an amino acid or peptide bearing an additional basic nitrogen functionality, a metal-chelating clearing agent, and an antibody or antibody fragment directed to an antigen on an antibody or antibody fragment which specifically binds to a tumor-associated antigen. In even more preferred embodiments, the clearing agent or agents are selected from the group consisting of lysine, polylysine, DOTA, DTPA, PLED, EDTA, and anti-idiotypic Fabxe2x80x2 fragments. In especially preferred embodiments, the clearing agent is selected from the group consisting of lysine, polylysine, and galactosylated anti-idiotypic Fabxe2x80x2 fragments. In still more preferred embodiments, the galactosylated anti-idiotypic Fabxe2x80x2 fragments are humanized or chimeric Fabxe2x80x2 fragments.
In other preferred embodiments, a method for treating a patient includes administering a radioimmunoconjugate of the present invention to a patient and subsequently grafting bone marrow or autologous stem cells to the patient.
In still other preferred embodiments, a method for treating a patient includes administering a radioimmunoconjugate of the present invention to a patient in conjunction with a clearing agent as described above and subsequently grafting bone marrow or autologous stem cells to the patient.
Other preferred embodiments of the invention provide a kit for use in radioimmunotherapy, comprising a molecule with a radioisotope binding site linked to or on an antigen-binding fragment of an antibody which specifically binds to a tumor-associated antigen, and at least one clearing agent.
More preferred embodiments of the invention provide a kit for use in radioimmunotherapy as described above wherein the clearing agent is selected from the group consisting of chelating clearing agents, an antibody or antibody fragment directed to an antigen on an antibody or antibody fragment which specitically binds to a tumor associated antigen, and clearing agents which prevent reuptake of small molecules and ions in the tubules of the kidney.
Other preferred embodiment of the invention provide a kit for use in radioimmunotherapy as described above wherein the at least one clearing agent is selected from the group consisting of DTPA, DOTA, EDTA, PLED, D-lysine, polylysine, and anti-idiotypic antibody Fabxe2x80x2 fragments. In especially preferred embodiments, the kit provides at least one clearing agent, wherein the clearing agent is an anti-idiotypic galactosylated Fabxe2x80x2 fragment, and in other even more preferred embodiments the anti-idiotypic galactosylated Fabxe2x80x2 fragment is a chimeric or humanized Fabxe2x80x2 fragment.
Additional aspects, features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The embodiments and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
Generally, the present invention provides a radioimmunoconjugate directed to a diseased tissue target. The radioimmunoconjugates of the present invention comprise a cytotoxic radioisotope bound to a binding site linked to or on an antigen-binding fragment of an antibody which specifically binds to a tumor-associated antigen.
Generally, the present invention also provides a method for treating a patient comprising administering a radioimmunoconjugate of the present invention to a patient. Preferably, the radioimmunoconjugate is administered to a patient in conjunction with one or more clearing agents to prevent unbound radioisotope from accumulating in and damaging organs and tissues in the body. Another preferred method of treating a patient comprises administering a radioimmunoconjugate to a patient and subsequently grafting bone marrow or autologous stem cells to the patient. A most preferred method of treatment comprises administering a radioimmunoconjugate of the present invention to a patientin conjunction with one or more clearing agents and subsequently grafting bone marrow or autologous stem cells to the patient.
The radioisotopes of the present invention include both alpha and beta emitting radioisotopes. Preferred radioisotopes of the present invention include alpha-emitting radioisotopes. Especially preferred radioisotopes of the present invention include 213Bi, 90Y, and 211At.
The radioimmunoconjugates of the present invention include a binding site linked to or on an antigen-binding fragment of an antibody which specifically binds to a tumor-associated antigen. Preferably, the antibody is an IgG immunoglobulin. However, it should be noted that the fragment of an antibody can be a fragment of any of the immunoglobulin types that binds to an antigen on the surface of diseased tissue or cancer cells. The antigen-binding fragment of the antibody which specifically binds to a tumor-associated antigen is preferably a Fabxe2x80x2 fragment. Especially preferred are Fabxe2x80x2 fragments which are humanized or chimeric in nature such that immunogenic response is minimized. Humanized and chimeric Fabxe2x80x2 fragments may be prepared by methods known to those skilled in the art.
The binding site attached to a fragment of an antibody includes groups that chelate and those that are covalently bonded to a radioisotope of the present invention. The binding site of the present invention thus includes the sulfhydryl (SH) group on the cysteine residue on Fabxe2x80x2 fragments after cleavage of the disulfide linkage. The sulfhydryl group will be found to bind many radoimetal cations including various isotopes of lead and bismuth. The binding site also includes groups such as DTPA, DOTA, PLED, EDTA, crown ethers, cryptands, various phosphines including, but not limited to, diphosphines and triphosphines, thiols and any other chelating group that is known by those skilled in the art to bind the radioisotopes of the present invention. Preferred binding sites include DTPA and DOTA.
As described above, the term binding site as used herein is broad enough to encompass groups that are covalently attached to a radioisotope. This is particularly relevant to radioisotopes such as 211At which forms covalent bonds to various classes of organic compounds e.g. toluenes, phenols and phenol ethers (e.g. tyrosine, 4-methoxyphenylalanine, and anisole), benzene, anilines, imidazoles, phenylalanine, and pyrimidines among others. Thus, the term binding site attached to a fragment of an antibody in this context refers to the group to which the astatine isotope is covalently attached.
One or more clearing agents can be used to practice the method of the invention. Clearing agents in this context serve several different purposes. Non-targeted antibody fragment conjugate advantageously is cleared from circulation after sufficient time has elapsed for it to accrete at the target site. The clearing agent can be an antibody that specifically binds to any part of the conjugate. To the extent that the therapeutic radioisotope becomes detached from its binding site, usually a chelator, it advantageously is cleared with a chelating clearing agent. Glycosylation of clearing agents such as the foregoing can increase the rate of clearance by inducing rapid uptake by glycoside receptors in the liver. A further aid to clearance of non-targeted radioisotope can be achieved by the use of clearing agents that inhibit reuptake of small molecules in the kidney tubules. This mitigates radiation damage to the kidneys and accelerates clearance through the urinary bladder. It will be appreciated that combinations of these clearing agents advantageously are used to obtain the benefits each provides.
One type of clearing agent of the present invention includes metal-chelating agents that bind radioisotopes in the environment of the human body. Examples of this type of clearing agent include DTPA, DOTA, EDTA, PLED, and other materials known to those skilled in the art. Another type of clearing agent includes antibodies or antibody fragments directed against antiepitope on an antibody or antibody fragment which specifically binds to a tumor-associated antigen. Preferred clearing agents of this class include anti-idiotypic clearing agents in which the clearing agent is an antibody or antibody fragment directed to the paratope of the antibody or antibody fragment directed to an antigen on a tumor or cancer cell.
Other preferred clearing agents include galactosylated antibodies or antibody fragments which exhibit enhanced clearing ability. Suitable such agents include but are not limited to galactose-second-Ab-Fabxe2x80x2, where the second Ab binds to the targeting Ab. Other preferred clearing agents of this class include chimeric or humanized anti-idiotypic antibodies or antibody fragments, and particularly preferred clearing agents of this class are galactosylated humanized anti-idiotypic Fabxe2x80x2 fragments such as galactose-W12-Fabxe2x80x2.
Another type of clearing agent of the present invention prevents reuptake of small molecules and ions in the tubules of the kidney. Such clearing agents include both amino acids and peptides bearing a basic nitrogen functionality in addition to the xcex1-amino group found on naturally occurring amino acids. Thus, this class of clearing agents includes both enantiomers of lysine, ornithine, histidine, and arginine in addition to polymers such as polylysine. However, the D amino acids of lysine, ornithine, histidine, and arginine are preferred clearing agents of this type, and D-lysine is especially preferred. Also preferred, are polymers of lysine such as polylysine.
Because the three types of clearing agent work in different ways, they may be used together to enhance removal of undesired species from the system. Preferably, a clearing agent of the type that prevents reuptake of small molecules and ions in the tubules of the kidney is used in conjunction with a clearing agent of the type which is an antibody or fragment of an antibody directed to an antigen on the surface of the targeting antibody or antibody fragment. Especially preferred is the use of a galactosylated anti-idiotypic fragment of an antibody which is directed to the paratope on the targeting antibody or antibody fragment in conjunction with either enantiomer of lysine. Also preferred, is the use of a chelating clearing agent to facilitate removal of free radioisotope with the above clearing agent combinations.
The present invention also provides a kit for use in RAIT. The kits of the present invention include the uncomplexed radioimmunoconjugate of the present invention which can also be described as a molecule with a radioisotope binding site linked to or on an antigen-binding antibody fragment which specifically binds to a tumor-associated antigen. The kits also include at least one clearing agent as described above. Preferable clearing agents for use in the kit include those which prevent reuptake of small molecules and ions in the kidney tubules such as lysine and polylysine; those which chelate metals such as DOTA, DTPA, PLED, and EDTA; and those antibodies or antibody fragments, especially Fabxe2x80x2 fragments, which are directed to the molecule with a radioisotope binding site.
The kits of the present invention are intended to be used in the treatment of patients having diseased target tissue bearing the antigen to which the molecule with a radioisotope binding site specifically binds. Thus, the molecule with the radioisotope binding site can be mixed with a radioisotope such as 213Bi or 90Y to prepare a radioimmunoconjugate of the present invention. The radioimmunoconjugate may then be injected into the patient followed by injection of the clearing agent to minimize damage to bone marrow, liver, kidneys, and other organs. A clearing agent that prevents the reuptake of small molecules in the kidney tubules may be injected prior to, with, or after injection of the radioimmunoconjugate. In this manner, the targeted tissue may be destroyed while minimizing damage to untargeted tissues or organs. Although the kits of the present invention include at least one clearing agent, the kits may contain other clearing agents. For example, a preferred kit of the present may include a chimeric or humanized galactosylated anti-idiotypic clearing agent and lysine to be used in conjunction for minimizing damage to non-targeted tissue or organs and additionally preventing reuptake in the kidney tubules.
The present invention may be embodied in other specific forms without departing from its spirit or its central characteristics, The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims, rather than by the foregoing or following description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.
Experiments were conducted to examine the efficacy of the radioimmunoconjugates of the present invention as therapeutic agents in the treatment of cancer and diseased tissue. These studies found that radioimmunoconjugates of both 213Bi and 90Y were highly effective in treating tumors induced by intrasplenic injection of human colon cancer cell line, GW-39, in mice.
A murine monoclonal antibody CO17-1A, an IgG2a isotype, was chosen as the immunoglobulin for the experiments. It is directed against a 41 kD glycoprotein found on human gastrointestinal malignancies. CO17-1A has an affinity constant of 5xc3x97107 1/mol to its antigen. After binding to the antigen. CO17-1A is readily internalized into the antigen-expressing tumor cells. The anti-human-CD3 antibody, OKT3, was used as irrelevant isotype-matched control. OKT3 is also a murine IgG2a isotype and was obtained from CILAG (Sulzbach/Taunus, Germany).
Tumors were induced in female nude mice by injecting them with 200 xcexcL of a 20 percent cell suspension of human carcinoma cell line, GW-39 serially propagated by preparing a mince through a 40 mesh screen and rinsing with Hank""s balanced salt solution. After waiting approximately 10 days, tumors reached a size of 100-200 mg, and the mice were then used in the experiment.
Fabxe2x80x2 fragments of CO17-1A were prepared from the complete immunoglobulin by pepsin digestion followed by subsequent disulfide reduction with cysteine. After purification and blocking with iodoacetamide, the Fabxe2x80x2 fragments were reacted with isothiocyanate benzyl-DTPA to prepare the CO17-1A DTPA-Bz-Fabxe2x80x2 conjugate. After purification, the DTPA-Bz-Fabxe2x80x2 fragments were separately reacted with 213Bi, 90Y, and 88Y salts to produce the radioimmunoconjugates of the present invention, and the 88Y complex was used to analyze biodistribution. The prepared radioimmunoconjugates were injected into the mice within 20 minutes of their final preparation.
The biodistribution of bismuth versus yttrium-containing CO17-1A Fabxe2x80x2 were compared to free Bi3+ or Y3+ in tumor, blood, kidneys and bone of subcutaneous GW-39 tumor-bearing nude mice. Due to the short physical half-life of 213Bi, biodistribution studies were performed up to 5 hours post injection in contrast to one week with yttrium-containing conjugates. No significant differences were observed between the biokinetics of 213Bi- or 90Y-containing immunoconjugates. The blood clearance was bi-exponential, with similar half-lives (txc2xd (xcex1)≈15 min, txc2xd (xcex2)≈5 hours) to those observed with radioiodinated Fabxe2x80x2 fragments. The uptake in the tumor was rapid, reaching its apogee as early as 1-4 hours post injection (approximately 4% ID/g). Renal accretion was predominant, with maximum uptake values of up to xe2x89xa780% ID/g. Application of D-lysine reduced the renal accretion four to five fold.
In contrast, it was shown that free bismuth exhibits a very high renal accretion (up to 150% ID/g), which cannot be blocked by lysine, whereas yttrium ions are well known to be bone-seeking. The comparison clearly shows in vivo stability of the conjugates compared to the free ions.
Maximum tolerated dose finding trials of 213Bi- and 90Y-containing CO17-1A Fabxe2x80x2 were undertaken. For this purpose, varying amounts of activity were injected starting at 100 xcexcCi and increasing in 10 to 20 percent steps. At each activity level, one group of animals (10 to 20 per group) received the bismuth or yttrium with no additional support, and a second group was treated with D-lysine. Blood counts (white blood cells and platelets), blood urea nitrogen (BUN) and creatinine as well as alkaline phosphatase and glutamate oxaloacetate transaminase were determined on the day of radioimmunoconjugate administration and at weekly intervals thereafter. Acute treatment-related death was defined as occurring within four weeks post RAIT, whereas later deaths were regarded as chronic toxicity, if these deaths could not be related to tumor growth.
700 xcexcCi of 213Bi-CO17-1A Fabxe2x80x2, and 250 xcexcCi of its 90Y counterpart were tolerated by all animals without acute treatment-related lethality, regardless of lysine administration. A 10% increase of these activities resulted in an at least 10% lethality. The blood cell counts and BUN levels at these respective maximum tolerated activities for 213Bi- and 90Y-Fabxe2x80x2, respectively, were determined. The nadirs of leukocyte and thrombocyte counts were reached at 1 week after radio-antibody administration, and at both respective maximum tolerated activities, the severity of myelotoxicity was not significantly different, regardless of the radionuclide. The recovery from myelotoxicity, however, appeared faster with the shorter-lived 213Bi than with the 90Y. Thus, with 213Bi-Fabxe2x80x2 the time to complete recovery was two to three weeks post therapy while with 90Y-Fabxe2x80x2 the time to complete recovery was three to four weeks.
No significant differences with respect to the severity of acute myelotoxicity were observed whether the animals were given D-lysine or not. However, at a dose level of 300 xcexcCi of 90Y-Fabxe2x80x2, all animals without kidney protection died as compared to a 50% mortality when they were treated with D-lysine. In contrast, myelotoxicity-related lethality rates were similar at 800 (15% lethality) and 900 xcexcCi (35% deaths) of 213Bi-Fabxe2x80x2 regardless of whether or not lysine was given.
In contrast, profound differences with respect to chronic toxicity were observed between animals given lysine and those not given lysine. The data show that in non-lysine treated animals, after a transient (2-3 weeks) episode of BUN elevation with subsequent normalization, BUN levels began to rise at six to seven weeks after therapy. The serum creatinine level followed the BUN value, but was less sensitive than the latter. The development of chronic renal failure was more dramatic (more steeply rising BUN levels in the 90Y than in the 213Bi-treated groups. All animals treated with 250 xcexcCi 90Y-Fabxe2x80x2 without lysine protection died within three months after therapy, whereas the lethality of animals given 700 xcexcCi of 213Bi-Fabxe2x80x2 was only 15% after 5 months. Lysine treated groups survived without any signs of renal compromise, and there was no induction of renal insufficiency even six months after treatment. When combining kidney protection by lysine with bone marrow transplantation, the animals survived 400 xcexcCi of 90Y- and 1100 xcexcCi of 213Bi-CO17-1A-Fabxe2x80x2, whereas 10% higher activities again led to an at least 10% lethality.
The therapeutic effect of 213Bi-versus 90Y-containing CO17-1A-Fabxe2x80x2 at their respective maximum tolerated doses without or with bone marrow transplantation in subcutaneous GW-39 bearing animals also was studied. Both radioimmunoconjugates led to a significant growth retardation as compared to untreated controls or animals treated with the same activities of the Fabxe2x80x2 fragment of the irrelevant antibody OKT3. Anti-tumor effects improved with dose-intensification. However, at each maximum tolerated dose 213Bi-Fabxe2x80x2 was therapeutically superior to its 90Y-Fabxe2x80x2 analog, whereas no significant differences were observed between both radioisotopes with the irrelevant OKT3 Fabxe2x80x2.
The survival of animals bearing GW-39 liver metastases was determined. Groups of twenty animals each were left untreated, were given 250 xcexcCi of 90Y-CO17-1A or irrelevant OKT3-Fabxe2x80x2, or 700 xcexcCi 213Bi-CO17-1A or irrelevant OKT3-Fabxe2x80x2 at two weeks after tumor inoculation. Each of these groups was placed under kidney protection with D-lysine. Untreated animals died within six to eight weeks of rapidly progressing liver metastases. The survival of animals treated with the irrelevant antibody was prolonged only for one to three weeks, regardless of the radioisotope. In contrast, the mean survival of animals treated with 90Y-Fabxe2x80x2 was fifteen weeks, and twenty percent of mice treated in this manner survived for more than 30 weeks. Histology could not demonstrate any viable tumor cells in these long-term survivors. Thus, these animals were regarded as cured. The 95% cure rate of animals treated with 213Bi-CO17-1A-Fabxe2x80x2 was significantly higher than that for animals treated with its 90Y analog.
An external scintigraphic scan of two mice bearing multiple GW-39 liver metastases at one hour after the injection of 700 xcexcCi of 213Bi-CO17-1A-Fabxe2x80x2 without or with D-lysine administration shows the effect of lysine administration on the renal accretion of 213 Bi-CO17-1A-Fabxe2x80x2, and it shows good tumor uptake in multiple metastatic lesions in the liver.
The radiation dosimetry of 213Bi-versus 90Y-containing CO17-1A-Fabxe2x80x2 in subcutaneous GW-39 xerograft bearing nude mice, based on the biodistribution studies is summarized in Table 1. The model of Yoriyaz and Stabin was used since high energy xcex2-emitters like 90Y result in cross-organ irradiation. For the dosimetry of 90Y-CO17-1A-Fabxe2x80x2, the table accounts for cross-organ radiation, and for 213Bi-CO17-1A-Fabxe2x80x2, it differentiates into doses resulting merely from the self-absorption of the xcex1 particles of 213Po (first column) or including the xcex2 and xcex3 rays of 213Bi (with potential inter-organ crossfire, second column).
As a result of tumor growth-retardation being the major observed effect in all treated groups, the correlation between mean tumor doses and the extent of induced growth retardation was an important measure of the activities of the radioimmunoconjugates. Since GW-39 is a very rapidly-growing cell line with tumor-volume doubling times of less than one week, the correlation between tumor doses and the mean time needed for quadruplication of tumor volume was analyzed. With 90Y-Fabxe2x80x2, below a tumor dose of approximately 5 Gy, no significant tumor growth delay was noticeable. Above the threshold, tumor growth was retarded in a dose-dependent fashion up to seven-fold at approximately 20 Gy. The resulting non-linear regression curve (regression coefficient: r=0.96) resembles a shoulder curve with its shoulder at approximately 5 Gy. In distinct contrast, no threshold dose was seen with the 213Bi-CO17-1A-Fabxe2x80x2, and the time to tumor volume quadruplication was prolonged in an almost linearly increasing manner with increasing tumor dose (regression coefficient: r=0.98). Comparable anti-tumor efficacy was seen with 213Bi-CO17-1A-Fabxe2x80x2 at doses which were approximately half as high as those needed with the 90Y radioisotope.